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3-D Culturing Prompts ESC Aggregates to Spontaneously Form Optic Cup

Two ES cell-derived optic cup formed by self-organization in 3D culture: Green color is fluorescence of GFP protein that was engineered to mark retinal tissue. [M. Eiraku and Y.Sasai at RIKEN Center for Developmental Biology]

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Scientists have demonstrated for the first time the ability of cultured stem cells to spontaneously differentiate and develop into the 3-d structure of the embryonic eye. The researchers, led by a team at the Riken Center for Developmental Biology in Japan, based their culture system on an ES cell culture medium that had previously been used to differentiate embryonic stem cells into a range of neuronal cell types including structurally organized cerebral cortical neurons.

When they grew clusters of mouse embryonic stem cells in a 3-D culture medium modified for their purpose, the cells organized into two-layered optic-cup-like structures within 10 days in the absence of any external signaling source such as a lens or surface ectodermal tissues. The pigmented and neuronal character of the outer and inner layers of cells in the spontaneously formed tissues were confirmed by gene expression. The work, led by Mototsugu Eiraku, Ph.D., deputy leader of the Four-Dimensional Tissue Analysis Unit at Riken, is detailed in Nature in a paper titled “Self-organizing optic-cup morphogenesis in three-dimensional culture.”

Dr. Eiraku’s team had previously demonstrated the self-formation of stratified cerebral cortical tissues in culture, where floating aggregates of ES cells were cultured under low growth-factor conditions (serum-free floating culture of embryoid-body-like aggregates with quick reaggregation, or SFEBq). The same approach had also been used to induce retinal differentiation in a modified SFEBq culture using transient activin treatment, but in this instance no clear formation of retinal epithelial structures was observed. For their latest attempt the team further modified the culture medium and added basement-membrane matrix components to promote the formation of rigid continuous epithelial structure.

They were able to follow the process of morphogenesis in 3-D, using a specially assembled multi-photon live-imaging system in combination with a full-sized CO2/O2 incubator. The invagination process consisted of four consecutive phases consistent with in vivo development, they report. On day 6, the evaginated vesicle was hemispherical in shape (phase 1). In phase 2, occurring around day 7, the distal portion of the vesicle became flattened.

Subsequently, in phase 3, the angle at the joint (hinge) between the neural retina and RPE domains became narrower or even acute. Then, on day 8, the neural retina epithelium started to expand as an apically convex structure, forming a cup via progressive invagination (phase 4).

When the researchers examined cytoskeletal features, they found that myosin activity dropped in the region of the epithelium that bent inwards to form the cup, providing the flexibility needed to form a pocket that was driven by expansion of the epithelium through cell division. Moreover, when the neuronal layer from the optic cup was removed and allowed to develop in 3-D cell culture conditions optimized for neuronal maturation, the retinal neurons underwent mitosis and organized inot a six-layer stratified and synapse-forming neuronal structure that closely resembles the post-natal retina.

The self-directed organization of the complex optic-cup and neural retina morphology was unexpected as the ESC culture actually started as patternless aggregates of homogeneous pluripotent cells and continued under a uniform culture environment, the authors admit. Nevertheless, they suggest, the self-formation of fully stratified 3-D neural retina tissues “heralds the next-generation of generative medicine in retinal degeneration therapeutics and opens up new avenues for the transplantation of artificial retinal tissue sheets, rather than simple cell grafting.”

Co-author Yoshiki Sasai, Ph.D., comments, “What we’ve been able to do in this study is resolve a nearly century-old problem in embryology by showing that retinal precursors have the inherent ability to give rise to the complex structure of the optic cup. It’s exciting to think that we are now well on the way to becoming able to generate not only differentiated cell types but organized tissues from ES and iPS cells, which may open new avenues towards applications in regenerative medicine.”

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